Diffraction anomaly sensor having grating coated with protective dielectric layer

ABSTRACT

A method and apparatus for optically assaying a targeted substance in a sample using a diffraction anomaly grating sensor. The optical sensor has a diffraction grating coated with at least one dielectric layer such that the sensor is sensitized to interact with the targeted substance. Upon interaction, light incident upon the sensor at a particular angle propagates through the dielectric, thereby exhibiting a dip in zero-order reflectance. Advantages of the present invention include facilitating increased sensitivity while protecting the metal grating from tarnishing and degradation. The present invention also allows for the construction of sensors that are sensitized to a plurality of substances, thus eliminating the need for an operator to reconfigure the sensing system in order to assay different substances.

FIELD OF THE INVENTION

This invention relates generally to the field of optical sensing and,more particularly, to a method and apparatus for assaying chemical andbiological materials.

BACKGROUND OF THE INVENTION

Recently, extremely sensitive optical sensors have been constructed byexploiting an effect known as surface plasmon resonance (SPR). Thesesensors are capable of detecting the presence of a wide variety ofmaterials in concentrations as low as picomoles per liter. SPR sensorshave been constructed to detect many biomolecules includingdinitrophenyl, keyhole limpet hemocyanin, α-Feto protein, IgE, IgG,bovine and human serum albumin, glucose, urea, avidin, lectin, DNA, RNA,hapten, HIV antibodies, human transferrin, and chymotrypsinogen.Additionally, SPR sensors have been built which detect chemicals such aspolyazulene and various gases including halothane, tricloroethane andcarbon tetrachloride.

An SPR sensor is constructed by sensitizing a surface of a substrate toa specific substance. Typically, the surface of the substrate is coatedwith a thin film of metal such as silver, gold or aluminum. Next asensitizing layer, such as a monomolecular layer of complementaryantigens, is covalently bonded to the surface of the thin film. In thismanner, the thin film is capable of interacting with a predeterminedchemical, biochemical or biological substance. When an SPR sensor isexposed to a sample that includes the targeted substance, the substanceattaches to the sensitizing layer and changes the effective index ofrefraction at the surface of the sensor. Detection of the targetedsubstance is accomplished by observing the optical properties of thesurface of the SPR sensor.

The most common SPR sensor involves exposing the surface of the sensorto a light beam through a glass prism. At a specific angle of incidence,known as the resonance angle, a component of the light beam's wavevectorin the plane of the sensor surface matches a wavevector of a surfaceplasmon in the thin film, resulting in very efficient energy transferand excitation of the surface plasmon in the thin film. As a result, atthe resonance angle the reflected light from the surface of the sensorexhibits a sharp dip that is readily detected. When the targetedsubstance attaches to the surface of the sensor, a shift in theresonance angle occurs due to the change in the refractive index at thesurface of the sensor. A quantitative measure of the concentration ofthe targeted substance can be calculated according to the magnitude ofshift in the resonance angle.

SPR sensors have also been constructed using metallized diffractiongratings instead of prisms. For SPR grating sensors, resonance occurswhen a component of the incident light polarization is perpendicular tothe groove direction of the grating and the angle of incidence isappropriate for energy transfer and excitation of the thin metal film.As with prism-based sensors, a change in the amount of light reflectedis observed when the angle of incidence equals the resonance angle.Previous SPR grating sensors have incorporated square-wave or sinusoidalgroove profiles.

SPR grating sensors offer many benefits over SPR sensors having glassprisms including a thicker, more robust metal film. Furthermore, thegrating period for an SPR grating sensor can be adjusted for any desiredresonance angle. Despite these benefits, both current SPR gratingsensors and prism-based sensors are susceptible to degradation due tooxidation of the metal film and its continuous exposure to the sample.For this reason, the thin metal film is usually constructed with a metalthat tarnishes slowly, such as gold, rather than a highly sensitivemetal which tarnishes more quickly, such as silver. Another disadvantageof current SPR sensors is that the metal film causes many biologicalsubstances to denature, thus leading to erroneous readings. For thereasons stated above, and for other reasons stated below which willbecome apparent to those skilled in the art upon understanding thepresent invention, there is a need in the art for an optical sensorhaving improved sensitivity and less susceptibility to degradation.

SUMMARY OF THE INVENTION

In one aspect, the invention is a sensor for assaying a substance in asample. The sensor comprises a substrate having a plurality of groovesin a surface. The grooves are formed in a substantially periodic profilesuch as sinusoidal, trapezoidal or any other suitable formation. A metallayer is formed outwardly from the surface of the substrate. In thismanner, the metal layer substantially conforms to the grooved profile ofthe surface of the substrate. A dielectric layer is formed outwardlyfrom the metal layer and is selected so as to suppress the zero-orderreflectance of light polarized parallel to the grooves of the surfacefor at least one angle of incidence. It is preferable that thedielectric layer has a thickness of at least 50 nm or, more preferably,at least 130 nm.

The sensor may include a sensitizing layer formed outwardly from thedielectric layer. The sensitizing layer is capable of interacting withthe substance in the sample and thereby changes the angle of incidenceat which the sensor suppresses the zero-order reflectance of incidentlight. In one embodiment the sensitizing layer comprises a layer ofantigens. In another embodiment, the dielectric layer is capable ofinteracting with the substance in the sample.

In yet another embodiment, the invention is a sensor for assaying aplurality of substances in a sample. The sensor includes a substratehaving a surface formed in a substantially periodic grooved profile anda metal layer is formed outwardly from the surface of the substrate. Aplurality of substantially non-overlapping dielectric layers are formedoutwardly from the metal layer. Each of the dielectric layers is capableof suppressing the zero-order reflectance of incident light for at leastone corresponding angle of incidence and polarization. This embodimentfacilitates the sensing of a plurality of substances in a sample or thereuse of a single sensor for multiple samples having various substances.

In one aspect, the invention is a method for assaying a substance in asample using a sensor having a grooved diffraction grating surfacecoated with a dielectric layer. The sensor is exposed over a pluralityof incident angles by a light beam polarized parallel to the grooves inthe diffraction grating surface. A controller detects a firstdiffraction anomaly angle at which zero-order reflectance of a componentof the incident light having a polarization parallel to the grooves ofthe sensor is a minimum. After interacting the sensor with the sample,the sensor is exposed a second time with a light beam over the pluralityof incident angles and a second diffraction anomaly angle is detected. Ameasure of the substance in the sample is determined as a function ofthe first diffraction anomaly angle and the second diffraction anomalyangle.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic side view of one embodiment of a sensing systemthat detects a substance by exposing a diffraction anomaly sensor havinga dielectric coated metal grating and detecting a shift in the incidenceangle that results in minimum reflectivity;

FIG. 2 is a schematic side view of one embodiment of a diffractionanomaly sensor having a dielectric-coated metal grating in accordancewith the present invention;

FIG. 3 is a graph that illustrates the calculated zero-order reflectanceover angles of incidence ranging from 0° to 20° for several diffractionanomaly sensors having dielectric layers of differing thickness; and

FIG. 4 is a schematic side view of one embodiment of a diffractionanomaly sensor having a plurality of dielectric layers, each dielectriclayer substantially sensitized for a different targeted substance.

DETAILED DESCRIPTION

In the following detailed description, references are made to theaccompanying drawings that illustrate specific embodiments in which theinvention may be practiced. Electrical, mechanical and structuralchanges may be made to the embodiments without departing from the spiritand scope of the present invention. The following detailed descriptionis, therefore, not to be taken in a limiting sense and the scope of thepresent invention is defined by the appended claims and theirequivalents.

FIG. 1 illustrates a sensing system 10 in accordance with the presentinvention. Sensing system 10 includes light source 20, diffractionanomaly sensor 50, polarizing beamsplitter 80, detector 60 and detector65. Light source 20, such as a laser, produces a light beam 25 incidentupon sensor 50. Sensor 50 reflects light beam 25 as light beam 70 ontopolarizing beamsplitter 80. Polarizing beamsplitter 80 splits light beam70 into component 85 and component 90 which are incident upon detectorarray 60 and detector array 65, respectively.

Sensor 50 is a diffraction anomaly sensor having a metal grating that iscoated with a dielectric layer as discussed in detail below. Similar toSPR sensors, sensor 50 exhibits a change in reflectivity, referred to asa diffraction anomaly, when exposed with light beam 25 at a particularangle of incidence. Unlike an SPR sensor, however, the change inreflectivity of sensor 50 occurs for light polarized parallel to thegrating grooves rather than perpendicular to the grating grooves. Inaccordance with the present invention, it is observed that the effectiveindex of refraction at the surface of sensor 50 changes in a mannersimilar to an SPR sensor when sensor 50 is smeared with a samplecontaining a targeted substance. It is further observed that the changein the index of refraction in turn shifts the incidence angle at whichthe diffraction anomaly occurs. Furthermore, for a fixed wavelength oflight beam 25, the diffraction anomaly angle is strongly dependent uponthe amount of targeted substance present in the sample. In this manner,sensor 50 exhibits a shift in the anomaly angle that is comparable to aSPR sensor, even though the metal grating of sensor 50 is coated with adielectric layer. Therefore, a quantitative measure of the targetedsubstance can be calculated by measuring the resulting shift in theanomaly angle.

After exposing sensor 50 to the sample, the new anomaly angle for sensor50 is determined by directing light beam 25 to sensor 50 over a range ofincidence angles. In one embodiment, sensor 50 is rotated so as to varythe angle of incidence of light beam 25. In another embodiment, sensor50 is fixed and light source 20 directs light beam 25 to sensor 50 overa range of incidence angles.

Polarizing beamsplitter 80 splits light beam 70 such that component 85has a wave vector parallel to the grooves of the surface of sensor 50and component 90 has a wave vector perpendicular to the grooves of thesurface of sensor 50. A controller (not shown) monitors detectors 60 and65 and continuously ratios the intensities of light component 85 andlight component 90 received by detectors 60 and 65. In this manner,light fluctuations of the light source, or other system variations suchas ripples in the sample, do not affect the calculation of the targetedspecies in the sample. Based on the calculated ratio for each sensingelement for detector arrays 60 and 65, the controller determines a newdiffraction anomaly angle and calculates a measure of the targetedsubstance in the sample based on the new diffraction anomaly angle. Inanother embodiment, the controller monitors the diffraction anomalyangle and sounds an alarm when the calculated measure of the targetedsubstance exceeds a predetermined threshold. After sensing is complete,SPR grating sensor 50 may be disposed or may be washed and reused.

FIG. 2 illustrates in detail one embodiment of diffraction anomalysensor 50 in accordance with the present invention. Sensor 50 includessubstrate 100 having a grooved surface 105. The grooves of surface 105are periodic and, in one embodiment, have a square cross-sectionalshape. Other cross-sectional shapes are contemplated for the grooves ofsurface 105 including but not limited to sinusoidal, trapezoidal andtriangular. The period of the grooves of surface 105 may range from lessthan 0.4 μm to over 2.0 μm.

Thin metal layer 110 is formed outwardly from surface 105 of substrate100 and comprises any suitable metal such as aluminum, gold or silver.Dielectric layer 120 is formed outwardly from metal layer 110 andthereby protects metal layer 110 from oxidation and general degradation.In this manner, metal layer 110 may comprise any suitable metal and maybe selected to optimize sensitivity. In one embodiment, layer 110comprises silver having a thickness of approximately 100 nm. As isdescribed in detail below, the diffraction anomaly exhibited by sensor50 is directly affected by the thickness and dielectric constant ofdielectric layer 120. Dielectric layer 120 comprises any suitabledielectric material such as silicon nitride, Si₃N₄, and is preferably atleast 50 nm thick. Alternatively, dielectric layer 120 may have athickness of at least 100 nm or, more preferably, at least 130 nm.

In one embodiment, sensitizing layer 130 is formed outwardly fromdielectric layer 120. Because dielectric layer 120 is disposed betweensensitizing layer 130 and metal layer 110, dielectric layer 120 preventschemical reaction between the metal layer 110 and sensitizing layer 130.Sensitizing layer 130 is selected to interact with a predeterminedchemical, biochemical or biological substance 140 contained in thesample. In this manner, sensitizing layer 130 may comprises a layer ofantigens capable of trapping a complementary antibody. Recently, severaltechniques have been developed for attaching antigens to dielectriclayer 120 such as by spin coating with a porous silica sol-gel or ahydrogel matrix. Preferably, sensitizing layer 130 is less than 100 nmthick. In another embodiment, dielectric layer 120 is selected so as tointeract directly with substance 140, thereby eliminating the need forsensitizing layer 130.

Unlike conventional SPR sensors, diffraction anomaly sensor 50 exhibitsa reflectance dip for light polarized parallel to the grooves of surface105. When light beam 25 has an angle of incidence equal to thediffraction anomaly angle for sensor 50, light beam 70 is not receivedby detector 60 but propagates within dielectric layer 120. In thismanner, dielectric layer 120 acts as a waveguide and the dip inreflectivity is readily detected by the controller.

The following equations can be used to select dielectric layer 120 suchthat a diffraction anomaly angle, θ_(SP), occurs for component 85 havinga polarization parallel to the grooves of surface 105. Using aniterative process, a wave-vector for the diffraction anomaly resonance,k_(x), can be calculated from the following equation:${{\left( {{ɛ_{1}\sqrt{{ɛ_{1}k_{0}^{2}} - k_{x}^{2}}} + {ɛ_{0}\sqrt{{ɛ_{0}k_{0}^{2}} - k_{x}^{2}}}} \right)\left( {{ɛ_{2}\sqrt{{ɛ_{2}k_{0}^{2}} - k_{x}^{2}}} + {ɛ_{1}\sqrt{{ɛ_{1}k_{0}^{2}} - k_{x}^{2}}}} \right)} + {\left( {{ɛ_{1}\sqrt{{ɛ_{1}k_{0}^{2}} - k_{x}^{2}}} - {ɛ_{0}\sqrt{{ɛ_{0}k_{0}^{2}} - k_{x}^{2}}}} \right)\left( {{ɛ_{2}\sqrt{{ɛ_{2}k_{0}^{2}} - k_{x}^{2}}} - {ɛ_{1}\sqrt{{ɛ_{1}k_{0}^{2}} - k_{x}^{2}}}} \right){\exp \left( {2i\sqrt{{ɛ_{1}k_{0}^{2}} - {k_{x}^{2}d}}} \right)}}} = 0$

In this equation, ε₀ is the dielectric constant of the medium above thesubstrate, such as air or water, etc., ε₁ is the dielectric constant ofthe dielectric layer, and ε₂ is the dielectric constant of the metalfilm. Furthermore, k₀ is a wavevector of the incident light in vacuumand equals 2π/λ. Finally, d is the thickness of the dielectric layer.

Once the wavevector for the diffraction anomaly resonance has beenfound, the following equation can be used to solve for the diffractionanomaly angle, θ_(SP):${\sin \quad \theta_{SP}} = {{{- \left( \frac{m\quad \lambda}{n_{0}p} \right)}\cos \quad \varphi_{SP}} \pm {\sqrt{\left( \frac{k_{x}}{n_{0}k_{0}} \right)^{2} - \left( {\frac{m\quad \lambda}{n_{0}p}\sin \quad \varphi_{SP}} \right)^{2}}.}}$

In this equation, φ_(SP) is the azimuthal angle of incident light beam25 with respect to the grooves of surface 105, where 0° corresponds tothe plane of incidence perpendicular to the groove direction, n₀ is theindex of refraction of the sample, λ is the wavelength of light beam 25,p is the period of the grooves of surface 105, and m is an integer.Thus, a dielectric layer having a suitable dielectric constant may bereadily selected so as to suppress component 85 which has a polarizationparallel to the grooves in surface 105 of sensor 50.

FIG. 3 plots the calculated reflectance of sensor 50 over a range ofincidence angles for light beam 25. More specifically, the zero-orderreflectance of sensor 50 is plotted for incidence angles ranging from 0°(normal) to 20° and an azimuthal angle of 0°. Furthermore, reflectivityis plotted for various thicknesses of dielectric layer 120 of sensor 50.In modeling the reflectivity of sensor 50, a sinusoidal profile forsurface 105 was selected having a period of 0.66 μm and a peak-to-peakamplitude of 0.107 μm. Metal layer 110 was modeled by selectingoptically thick aluminum having an index of refraction of n=2+i8,assuming a wavelength of 780 nm for light beam 25. Furthermore,dielectric layer 120 was modeled by a dielectric having an index ofrefraction of n=2.

FIG. 3 illustrates that the diffraction anomaly angle shifts as thethickness of dielectric layer 120 is increased. In addition, FIG. 3 isrepresentative of the shifting of the diffraction anomaly angle ofsensor 50 due to interaction with the targeted species. Increasing thethickness of dielectric layer 120 represents the increase in thicknessof the sensitizing layer as the targeted substance attaches to it,resulting in a shift of the diffraction anomaly angle by approximately10 degrees.

As described above, dielectric layer 120 may be selected such that itinteracts with substance 140 directly, thereby eliminating the need forsensitizing layer 130. This technique is advantageous in that a sensormay be developed that is sensitized to a variety of differentsubstances. FIG. 4 illustrates one embodiment of a diffraction anomalysensor that is sensitized to interact with a plurality of substances.More specifically, sensor 200 includes substrate 200 having grooves withsquare cross-sectional profiles. Other cross-sectional profiles arecontemplated including sinusoidal, trapezoidal and triangular and thesquare groove profile is chosen for exemplary purposes only. The periodof the grooves in the surface of substrate 200 may range from less than0.4 μm to over 2.0 μm.

Thin metal layer 210 is formed outwardly from substrate 200 and issubstantially similar to thin metal layer 110 of FIG. 2. A plurality ofdielectric layers 220 ₁ through 220 _(N) are formed along metal layer210 and are substantially non-overlapping as shown in FIG. 4. Dielectriclayers 220 protect metal layer 210 from oxidation and degradation. Eachdielectric layer 220 is selected to interact with different chemical,biochemical or biological substance 140 contained in the sample. In thisconfiguration, sensor 200 is sensitized to interact with a variety ofsubstances. One advantage of this configuration is that an operator doesnot need to reconfigure sensing system 10 in order to assay differentsubstances. Another advantage is that a single sample may be assayed fora plurality of substances simply be interacting sensor 200 with thesample, selectively exposing dielectric layers 220 with an incidentlight beam and detecting any shift in the corresponding diffractionanomaly angle. The diffraction anomaly exhibited by sensor 200 isdirectly affected by the thickness and index of refraction of theexposed dielectric layer 220. In one embodiment, dielectric layers 220have substantially equal thickness of at least 120 nm. In anotherembodiment, the thickness of dielectric layers 220 varies in order tooptimize the sensitivity of sensor 200 to the corresponding substance140.

CONCLUSION

Various embodiments of a method and system for assaying a substance in asample using a diffraction anomaly sensor have been described. In oneembodiment, the present invention is a sensing system that exposes adiffraction anomaly sensor with a light beam and quantitatively measuresthe concentration of targeted substance by determining the correspondingdiffraction anomaly angle. In another embodiment, the present inventionis a diffraction anomaly sensor having a metal diffraction gratingcoated with a protective dielectric. For a particular angle ofincidence, incident light having a polarization component parallel tothe grooves within the metal grating propagates through the dielectriclayer causing a dip in zero-order reflectance. In another embodiment,the present invention is a diffraction anomaly sensor that is sensitizedto interact with a plurality of substances.

Several advantages of the present invention have been illustratedincluding eliminating the degradation and oxidation of the metal gratingof conventional SPR grating sensors. In this manner, the presentinvention allows the metal grating to be selected so as to optimize thesensitivity of the system. Furthermore, the present invention allows forthe construction of sensors that are sensitized to a plurality ofsubstances, thus eliminating the need for an operator to reconfigure thesensing system in order to assay different substances.

This application is intended to cover any adaptations or variations ofthe present invention. It is manifestly intended that this invention belimited only by the claims and equivalents thereof.

I claim:
 1. A sensor for assaying a substance in a sample comprising: asubstrate having a plurality of parallel grooves in a surface; a metallayer formed outwardly from the surface of the substrate, the metallayer substantially conforming to the grooved surface of the substrate;and a dielectric layer formed outwardly from the metal layer, thedielectric layer for suppressing reflection of incident light having apolarization parallel to the grooves of the substrate wherein for lighthaving a plane of incidence perpendicular to the grooves of thesubstrate the dielectric layer suppresses a component of the lighthaving a polarization parallel to the grooves of the substrate.
 2. Thesensor of claim 1, further comprising a sensitizing layer formedoutwardly from the dielectric layer, wherein the sensitizing layerinteracts with the substance in the sample.
 3. The sensor of claim 1,wherein the dielectric layer interacts with the substance in the sample.4. The sensor of claim 1, wherein the dielectric layer has a thicknessof at least 50 nm.
 5. The sensor of claim 1, wherein the dielectriclayer has a thickness of at least 130 nm.
 6. The sensor of claim 1,wherein a cross-sectional shape of the grooved surface of the substrateis substantially periodic.
 7. The sensor of claim 6, wherein thecross-sectional shape of the grooved surface is selected from the set ofsinusoidal, trapezoidal and triangular.
 8. The sensor of claim 2,wherein the sensitizing layer comprises a layer of antigens.
 9. A sensorfor assaying a plurality of substances in a sample comprising: asubstrate having a plurality of parallel grooves in a surface; a metallayer formed outwardly from the surface of the substrate, the metallayer substantially conforming to the grooved surface of the substrate;and a plurality of substantially non-overlapping dielectric layersformed along the metal layer for suppressing reflectance of an incidentlight beam having a polarization component parallel to the grooves ofthe substrate and a plane of incidence perpendicular to the grooves ofthe substrate.
 10. The sensor of claim 9, wherein each dielectric layerinteracts with at least one of the plurality of substances in thesample.
 11. The sensor of claim 9, further comprising a sensitizinglayer formed outwardly from the plurality of dielectric layers, thesensitizing layer capable of interacting with the substance in thesample.
 12. The sensor of claim 9, wherein each of the dielectric layershas a thickness of at least 50 nm.
 13. The sensor of claim 9, whereineach of the dielectric layers has a thickness of at least 130 nm. 14.The sensor of claim 9, wherein a cross-sectional shape of the groovedsurface of the substrate is substantially periodic.
 15. The sensor ofclaim 14, wherein the cross-sectional shape of the grooved surface isselected from the set of sinusoidal, trapezoidal and triangular.
 16. Thesensor of claim 11, wherein the sensitizing layer comprises a layer ofantigens.
 17. A method for assaying a substance in a sample comprisingthe steps of: providing a sensor having a metal diffraction gratingcoated with a dielectric layer; emitting a light beam having apolarization component parallel to grooves in the metal diffractiongrating and incident within a plane perpendicular to the grooves in themetal diffraction grating; exposing the sensor with the light beam overa plurality of incident angles; detecting a first diffraction anomalyangle during the first exposing step at which zero-order reflectance ofthe incident light changes; interacting the sensor with the sample;exposing the sensor a second time with a light beam over the pluralityof incident angles; detecting a second diffraction anomaly angle duringthe second exposing step; and determining a measure of the substance inthe sample as a function of the first angle and the second angle. 18.The method of claim 17, further comprising the step of sounding an alarmwhen the determined measure of the substance in the sample exceeds apredetermined threshold.
 19. A method for assaying a substance in asample comprising the steps of: providing a sensor having a metaldiffraction grating having a plurality of grooves, wherein the metaldiffraction is coated with a dielectric layer; exposing the sensor witha light beam having a polarization component parallel to the grooves ofthe grating and incident within a plane perpendicular to the grooves inthe grating, and further wherein the component propagates substantiallywithin the dielectric layer when the sensor is exposed with the lightbeam at a diffraction anomaly angle; interacting the sensor with thesample; and determining a measure of the substance in the sample as afunction of a shift in the diffraction anomaly angle.
 20. The method ofclaim 19, wherein the exposing step comprises the step of polarizing thelight beam parallel to the grooves in the metal diffraction grating. 21.The method of claim 19, further comprising the step of sounding an alarmwhen the determined measure of the substance in the sample exceeds apredetermined threshold.
 22. The method of claim 19, wherein thedetermining step comprises the steps of: splitting a reflected lightbeam from the sensor into a first light beam having a polarizationvector parallel to grooves in the metal diffraction grating and a secondlight beam having a polarization vector perpendicular to grooves of themetal diffraction grating, wherein the first light beam and the secondlight beam each have a corresponding intensity; and monitoring a ratioof the intensities of the first light beam and the second light beam.23. A method for assaying a plurality of substances in a samplecomprising the steps of: providing a sensor having a metal diffractiongrating coated with a plurality of substantially non-overlappingdielectric layers, each dielectric layer for interacting with at leastone of the substances in the sample, wherein the diffraction grating hasa plurality of parallel grooves; selectively exposing at least one ofthe dielectric layers with a light beam at a corresponding diffractionanomaly angle and incident within a plane perpendicular to the groovessuch that a component of the light beam having a polarization parallelto the grooves in the sensor propagates substantially within the exposeddielectric layer; interacting the sensor with the sample; anddetermining a measure for each substance in the sample as a function ofa shift in the diffraction anomaly angle of the corresponding dielectriclayer capable of interacting with the substance.
 24. The method of claim23, further comprising the step of sounding an alarm when the determinedmeasure of each substance in the sample exceeds a corresponding one of aplurality of predetermined thresholds.
 25. A system for assaying asubstance in a sample, comprising: a sensor sensitized for interactingwith the substance in the sample comprising: a substrate having agrooved plurality of parallel grooves in a surface, a metal layer formedoutwardly from the surface of the substrate, the metal layersubstantially conforming to the grooved surface of the substrate, and adielectric layer formed outwardly from the metal layer; a light sourceexposing the sensor with a light beam at an angle of incidence having aplane of incidence perpendicular to the grooves in the surface of thesubstrate; a detector receiving light reflected from the sensor, thedetector responsive to light polarized parallel to the grooves in thesurface of the substrate; and a controller coupled to the detector forcalculating a measure of the substance in the sample as a function of ananomaly angle at which a change in zero-order reflectance of the lightbeam occurs.
 26. The system of claim 25, wherein the sensor furthercomprises a sensitizing layer formed outwardly from the dielectriclayer.
 27. The system of claim 25, wherein the dielectric layerinteracts with the substance in the sample.
 28. The system of claim 25,wherein the dielectric layer has a thickness of at least 50 nm.
 29. Thesystem of claim 25, wherein the dielectric layer has a thickness of atleast 130 nm.
 30. The system of claim 25, wherein a cross-sectionalshape of the grooved surface of the substrate is substantially periodic.31. The system of claim 30, wherein the cross-sectional shape of thegrooved surface is selected from the set of sinusoidal, trapezoidal andtriangular.
 32. The system of claim 26, wherein the sensitizing layercomprises a layer of antigens.
 33. The system of claim 25 wherein thedetector further comprises: a polarizing beamsplitter for receiving thereflected light and splitting the reflected light into a first componentand a second component, wherein the first component has a polarizationvector parallel to the grooves of the substrate and the second componenthas a polarization vector perpendicular to the grooves of the substrate;a first detector for receiving the first component of the reflectedlight, wherein the first detector has an output signal representative ofan intensity of the first component; and a second detector for receivingthe second component of the reflected light, wherein the second detectorhas an output signal representative of an intensity of the secondcomponent, wherein the controller ratios the output signal of the firstdetector and the output signal of the second detector, and furtherwherein the controller determines the anomaly angle according the ratioof the output signals.
 34. The system of claim 25 wherein the light beamhas a polarization parallel to the grooves in the surface of the sensor.